This invention relates to compositions and methods for controlling coleopteran insects by use of two proteins in combination which may be applied directly to the plant or produced thereon by microorganisms or by genetically modifying the plant to produce the proteins, to genes encoding these proteins, to methods for identifying such genes and proteins, and to recombinant microorganisms and plants capable of expressing these genes for use in controlling plant infestation by the target coleopteran insects.
The control of insect pests by naturally occurring proteins is a well established practice. The most commonly used insect control proteins are the endotoxins derived from Bacillus thuringiensis (B.t.) that are used to control both lepidopteran and coleopteran insect pests. Expression of these proteins in transgenic plants also confers protection against certain insect pests (Barton et al., 1987; Fischhoff et al., 1987 Perlak et al., 1990; Vaeck et al., 1987).
A variety of insect pests that cause significant economic losses were not previously known to be controlled by B.t. endotoxins. Boll weevil (BWV), Anthonomus grandis, corn rootworm (CRW), Diabrotica spp., and wireworm (WW), Melanotus spp. are examples of coleopteran insect pests that inflict significant crop damage yet, until recently were not known to be controlled by known B.t. endotoxins. Thus, it would be useful to identify new insecticidal proteins which, alone or in combination, are able to control these coleopteran insects. Furthermore, it would be useful to identify new insecticidal proteins with different modes of action to delay the development of B.t. endotoxin resistance in coleopteran pests such as the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say), that are currently controlled by certain B.t. endotoxins (Krieg et al., 1983).
Preparations of enzymes from several different sources are available from Sigma Chemical Company (St. Louis, Mo.) and other suppliers. Amino acid oxidases can also be obtained from sources including, but not limited to snake venom, mammalian, and avian sources (Bright and Porter, 1975). Lysine and other amino acid oxidases (E.C. 1.4.3.2) are naturally produced by micro-organisms such as Trichoderma sp., Neurospora sp., Penicillium sp., and Proteus sp. (Kusakabe et al., 1979; 1980; Niederman and Lerch, 1990; Knight, 1948; Stumpf and Green, 1944). Although lysine oxidase has been shown to have antitumor activity (Kusakabe et al., 1979; Id., 1980), there have been no reports of insecticidal activity associated with this enzyme. Also, there have been no reports of insecticidal activity being associated with an amino acid oxidase enzyme when combined with any other compound. However, we have unexpectedly found that a composition comprising a lysine oxidase and a previously unidentified Mr 50,000 protein yield potent insecticidal activity when combined in a mixture and ingested by an insect. The Mr 50,000 protein is described herein as a tedanalactam synthase shown herein to have at least one enzyme activity in which xcex941-piperideine-2-carboxylate is converted to tedanalactam. Described herein are methods for using a combination of lysine oxidase and tedanalactam synthase to control infestation of plants by insect pests.
This invention relates generally to novel compositions and methods for the control of undesired insects. It is therefore a particular object of the present invention to present materials and methods used in the preparation of compositions and plants capable of controlling insect infestation when ingested by the insect. It is also an object of the present invention to provide protein compositions capable of controlling BWV, CRW, WW, CPB or other insect pests, and genes useful in producing such proteins. It is a further object of the present invention to provide genetic constructs for and methods of inserting such genetic material into microorganisms and plant cells. It is another object of the present invention to provide transformed microorganisms and plants containing such genetic material. Still another object of this invention relates to methods and reagents such as polynucleotides and antibodies, and the use of such methods and reagents in kit form, for detecting the individual molecules which comprise the active compositions as noted herein. In addition, variants of the molecules which comprise the active compositions are also contemplated by this invention.
Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of a composition containing at least two proteins, lysine oxidase enzyme and tedanalactam synthase enzyme, which is capable of controlling insects, particularly coleopteran insects. These two proteins cause mortality and stunting of larvae of coleopteran insects when co-ingested. The proteins may be applied directly to plants or introduced in other ways such as through the application of plant-colonizing microorganisms or by transformed plants generated using recombinant DNA methods wherein the recombinant plants express genes encoding these enzymes.
In accomplishing the foregoing, there is provided, in accordance with one aspect of the present invention, a method of controlling insect infestation of plants comprising providing a composition containing at least a lysine or amino acid oxidase along with a second enzyme, which is preferably a tedanalactam synthase, for ingestion by the insect. It is apparent that neither protein alone is able to confer any insecticidal activity. However, it is the combination of the amino acid oxidase along with the second enzyme which is effective in confering insecticidal activity upon ingestion of such a composition by an insect. The composition, upon ingestion by the insect, contains a sufficient insecticidal amount of the proteins, such that the insect is unable to survive or is rendered incapable of causing further damage to a plant to which the composition has been applied. The composition contains a first enzyme which is a lysine oxidase enzyme, and a second enzyme which is capable of converting xcex941-piperideine-2-carboxylate to tedanalactam. The proteins in the composition are preferably isolated from extracts of fungal species fermentations in which the extracts have been shown to exhibit insecticidal activity. The fungal species herein which produces an insecticidally effective extract composition was determined to be a Trichoderma species of fungi, and in particular a Trichoderma viride. Another Monsanto Company fungal isolate was designated as Trichoderma sp. F22844 also produces an insecticidally effective extract composition. The genes encoding the proteins in the illustrative composition are therefore preferably isolated from a Trichoderma species of fungi, however, other uncharacterized fungal species are believed to contain at least a lysine oxidase gene and a second protein which, in combination provide efficacious insecticidal activity.
The composition can contain as the second enzyme a protein which is approximately 50,000 Da, which is also recognized by one skilled in the art as a protein or enzyme which is approximately Mr 50,000. It is believed that the second enzyme can be isolated from any number of species, however it is preferably isolated from a species which produces compounds which exhibit coleopteran insecticidal activity, and more preferably isolated from a fungal species. It is also believed that any fungal species which exhibits coleopteran insecticidal activity and also produces a lysine oxidase may also produce a second enzyme which in combination with the lysine oxidase confers effective insecticidal activity when ingested by target insect. Furthermore, any species which produces a lysine oxidase and which also contains a gene which hybridizes under stringent conditions to a Trichoderma species gene encoding an approximately Mr 50,000 Da protein which converts xcex941-piperideine-2-carboxylate to tedanalactam may confer effective insecticidal activity when a composition containing both enzymes is ingested by a target insect. The property of converting xcex941-piperideine-2-carboxylate to tedanalactam may be independent of the property which, in combination with an amino acid oxidase confers insecticidal activity upon the composition when ingested by the insect. It is intended that the composition not be limited to a combination of an amino acid oxidase and a tedanalactam synthase, but conceivably could also include the combination of a gene encoding an amino acid oxidase and a gene encoding a tedanalactam synthase, together with all necessary genetic regulatory elements required for expression, including repression and activation, transcription and translation, and post-transcriptional and post-translational modification signals incorporated therein. The genes as described could also be present either alone or in combination with each other on a single replicon.
The composition which confers coleopteran insecticidal activity is directed preferably to coleopteran species selected from the group consisting of Diabrotica species, Melanotus species, Leptinotarsa species, and Anthonomus species. Moreover, the composition is directed to controlling insects selected from the group consisting of boll weevil (BWV), corn rootworm (CRW), corn wireworm (WW), and the Colorado potato beetle (CPB).
The compositions in particular can contain the indicated enzymes in a mixture in which the molar ratios of the two enzymes are generally such that effective insect control is manifested. Insect control can be effected when the amino acid oxidase and the tedanalactam synthase are present within the composition in molar ratios of about 100:1 to about 1:1 respectively, or when the ratios are about 10:1 to about 1:1, respectively, or when the ratios of the two proteins are present from about 1:10 to about 1:1, or when the ratios of the two proteins are present from about 1:100 to about 1:1, respectively. In addition, effective concentrations of these proteins in a composition in which the proteins are each present from about one part per million to about 10 parts per million are effective in conferring insecticidal activity and control. The most effective insecticidal is activity is conferred when the proteins are each present in a composition from about one part per million to about 20 parts per million.
Another aspect of the present invention provides the structural genes which encode the enzymes which are the active components in the insecticidal compositions. Briefly, the genes can be isolated from genomic DNA and from cDNA molecules which are obtained by isolating MRNA from species which are shown to produce these enzymes. The structural genes encoding these enzymes, which may also be isolated as proenzymes or precursor proteins, preferably are identified by first isolating the active components or enzymes from extracts of organisms which produce these enzymes. Isolated enzymes can be digested with proteolytic enzymes, and amino acid sequences of proteolytic peptide fragments can be characterized. Redundant nucleotide probes corresponding to the characterized peptide fragments can be produced based on the deduced amino acid sequences, and used as probes or primers for identifying or amplifying particular segments of mRNA, cDNA, or genomic polynucleotides. Full length MRNA, full length CDNA, and uninterrupted full length genes can be further identified and isolated.
In accordance with other aspects of the present invention, there are provided methods and compositions for producing genetically transformed plants which express an amount of a lysine or other amino acid oxidase along with a second enzyme or tedanalactam synthase effective to control coleopteran insects. Recombinant plasmids have been produced which contain regulatory elements which function in plants for producing messenger RNA molecules, from which the proteins of the present invention are translated. Expression cassettes are disclosed which contain various elements alone or in combination for enabling the production of the amino acid oxidase or the tedanalactam synthase. Specifically, the amino acid oxidase gene is provided in a cassette comprising a polynucleotide sequence flanked 5xe2x80x2 by a promoter which functions in plants to cause the production of an RNA sequence is operably linked to an intron and a DNA sequence which functions in plants as a targeting signal or transit peptide and flanked 3xe2x80x2 by a DNA sequence which functions in plants to cause the addition of a 3xe2x80x2 non-translated polyadenylated nucleotide sequence to the 3xe2x80x2 end of the RNA is fused 3xe2x80x2 to the amino acid oxidase gene so that the expression of the cassette is under the control of the promoter. There are numerous alternatives to this construction, some of which are provided herein in specific examples. For example, the intron and targeting sequence can be replaced by a 5xe2x80x2 non-translated leader sequence; or the non-translated leader can be removed; the intron can be inserted between the non-translated leader and the oxidase gene or between the leader and the targeting sequence. The tedanalactam synthase can be assembled in a similar fashion, and specific examples are provided herein. An expression cassette for producing an amino acid oxidase can be combined into a single vector along with a cassette for producing a tedanalactam synthase so that delivery of both cassettes for simultaneous expression either in a plant or other organism such as a bacterium or fungi is also contemplated. Also, in a plant it is possible to express one of the cassettes in one tissue type, for example in roots, and express the other cassette in another tissue type, for example in leaves. It may also be possible to produce the proteins separately temporally or spatially, but in the same tissue type. For example, expression of one cassette in young leaves and the other cassette later in the same leaves is contemplated, however co-expression is normally desirable. The expression cassette can be designed to function in plants by using plant specific regulatory elements such as promoters, introns, targeting sequences, non-translated leaders, and 3xe2x80x2 polyadenylation sequences. The expression cassette can also be designed to function in prokaryotic systems as contemplated and described herein, also by using prokaryotic specific regulatory elements. The cassettes described herein can be inserted into plants by high velocity DNA coated particle projectile bombardment, by naked DNA protoplast transformation, or by bacterial mediated methods known in the art.
In describing this particular embodiment of the invention, it should be understood that expression of the amino acid oxidase, which can also be a precursor or proenzyme, and tedanalactam synthase can be controlled by two independent promoters from two separate and independent transcriptional units. It should also be understood that a single promoter could be used to drive expression of a single transcription unit containing an in frame translational fusion of both proteins. The hybrid polyprotein could then be post-translationally cleaved to yield both proteins by previously described schemes (Halpin and Ryan, WO 95/17514). Another advantage achieved by the present invention provides a peptide fusion to be produced from the genes encoding the two enzymes wherein the coding sequences of the two genes are fused in frame to allow for the expression of a recombinant gene encoding an in-frame translational peptide fusion of the amino acid oxidase and the tedanalactam synthase. The fusion can be one in which either enzyme is amino terminal with respect to the other. The fusion can be post-translationally cleaved by a plant endogenous endoprotease to produce an insecticidally active composition in the plant tissues so that lysine oxidase and tedanalactam synthase are present as separate and individual molecules. Alternatively, the fusion can be post-translationally cleaved by an endogenous insect endoprotease, generally found within the midgut of contemplated insect targets, so that the cleavage of the fusion protein produces an insecticidally active composition while within the midgut of the feeding insect.
In keeping with this aspect of the present invention, is the provision for a variety of promoters for transcriptional initiation and expression of the contemplated genes, in particular in plants. A number of promoters which are active in plant cells have been described in the literature. Such promoters may be obtained from plants or plant viruses and include, but are not limited to the nopaline synthase (NOS) and octopine synthase (OCS) promoters, which are carried on tumor-inducing plasmids generally found within virulent and non-virulent strains of Agrobacterium tumefaciens, the cauliflower mosaic virus (CaMV) 19S and 35S promoters, the light-inducible ribulose 1,5-bisphosphate carboxylase small subunit promoter(ssRUBISCO), and the Figwort Mosaic Virus 35S promoter (FMV). All of these promoters have been used to create various types of DNA constructs which have been expressed in plants (see for example Barry et al. U.S. Pat. No. 5,463,175, which is herein incorporated by reference). Particularly desirable promoters which are contemplated because of their constitutive nature are the Cauliflower Mosaic Virus 35S (CaMV35S) and the Figwort Mosaic Virus 35S (FMV35S) promoters which have previously been shown to produce high levels of expression in most plant organs. Other preferred promoters are root enhanced or root tissue specific promoters such as the Cauliflower Mosaic Virus derived AS4 promoter (also designated as the 4x as-1 or the 4as-1 promoter), the tobacco RB7 promoter, or the rice RC2 promoter (Lam et al., 1991; Yamamoto et al., 1991; Xu et al., 1995). The root enhanced or root tissue specific promoters would be particularly preferred for the control of corn rootworm (Diabrotica spp.) in transgenic corn plants. Other promoters are also contemplated which would direct tissue specific targeted expression are also contemplated, for example in tissue such as leaves, meristem, flower, fruit and organs of reproductive character. IN addition, chimeric promoters are also envisioned.
Other expression regulatory elements are considered to be of importance, especially in contemplation of transformed plant tissue or transformed plant cell expression. These elements comprise at least non-translated sequences and introns. For example, transcriptional events leading to RNA production from the contemplated DNA constructs as set forth herein could contain 5xe2x80x2 non-translated leader sequences. These sequences can be derived from new or existing promoters which are selected for gene expression, and can be specifically modified so as to increase translational efficiency of the mRNA. A plant gene leader sequence which has been shown to be particularly useful in the present invention is the petunia heat shock protein 70 leader (hsp70)(Winter et al., 1988). It has also been shown that introns are preferred for optimum expression in monocotyledenous plants. Any number of introns could function for this purpose, and without intending any limitation these could be selected from the group consisting of the maize hsp70 intron and the rice actin intron. Another nontranslated regulatory element of particular importance in plant systems are DNA sequences which function in plants to cause the addition of a 3xe2x80x2 non-translated polyadenylated nucleotide sequence to the 3xe2x80x2 end of an RNA sequence produced as a result of transcription from an indicated promoter sequence. These particular non-translated sequences are also commonly known as polyadenylation sequences or signals.
A further embodiment provides for the targeted delivery of a gene product to a particular organellar compartment, such as a vacuole, a mitochondrion, a chloroplast, a plastid, an endoplasmic reticulum compartment, a Golgi compartment, or even the nuclear or nucleolar domains. Particular peptide sequences have been shown to be necessary in obtaining efficient delivery of protein products to these sites, in particular signal peptides, signal sequences, and targeting sequences. Targeting signals or transit peptides contemplated herein, without intending to be limited to these, can be selected from the group consisting of a rice malate dehydrogenase amino terminal peroxisomal targeting signal and a maize ATP synthase beta subunit mitochondrial transit peptide.
A further embodiment of the present invention provides for the insertion of one or more of the contemplated expression cassettes along with any expression regulatory elements into the genome of a plant cell to form a stable recombinant plant cell. In one embodiment the DNA inserted into the plant genome would be comprised of a single cassette encoding a fusion peptide formed from the gene fusions described above, along with regulatory elements necessary for expression of the gene fusion. In another embodiment, the DNA inserted into the genome would be comprised of a single cassette encoding only an amino acid oxidase or a tedanalactam synthase, along with any regulatory elements necessary for expression of the gene. In the preferred embodiment, the DNA inserted into the plant genome would be comprised of a first gene cassette encoding an amino acid oxidase in which expression was controlled by a first promoter, and a second gene cassette encoding a tedanalactam synthase in which expression was controlled by a second promoter, along with any other necessary regulatory elements for expression of either gene. This embodiment contemplates that the first and the second promoters can be identical in sequence and function, or they can be different from each other, so long as each gene is expressed in an amount which provides a composition for controlling insect infestation of plants comprising a mixture of the enzymes when the mixture is ingested by a susceptible insect.
A further embodiment of the present invention provides methods for generating plants which express an insecticidally effective amount of a lysine or amino acid oxidase or proenzyme along with a tedanalactam synthase. The methods utilize contemplated DAN expression cassettes designed for producing either or both enzymes separately or in combination inserted into plasmids. The plasmid DNAs can be directly inserted into the genome of a plant by mechanical approaches, such as biolistic methods or by protoplast fusion techniques. A preferred method utilizes Agrobacterium mediated double border plant transformation, preferably using a DNA vector containing the desired expression cassette or cassettes flanked by Ti plasmid border recombination sequences in order to introduce the desired genes into the plant genome. The transformation procedure generally produces events which provides transformed plant cells selected on solid or in liquid media using any number of selectable markers known in the art, preferably glyphosate selectable markers such as GOX or EPSPS, antibiotic selectable markers, or others. Transformed cells obtained using these methods can be further regenerated to produce stably transformed genetically engineered plants which express insecticidally effective amounts of the amino acid oxidase or the tedanalactam synthase.
There is also provided, in accordance with another aspect of the present invention, transformed bacterial and plant cells that contain DNA comprised of the expressible gene cassettes as described above, along with appropriate control sequences necessary to provide for desired and appropriate expression of the coding sequences, producing insecticidally effective amounts of the enzymes. The control sequences can be any known in the art to function in a particular cell or organelle type. It is contemplated that the genes herein can be expressed in bacterial systems, plant nucleolar compartments, plant nuclear compartments, and in plant mitochondrial and chloroplast compartments.
While particular examples of using the invention described herein to control corn rootworm in corn or Colorado potato beetle in potato are provided, it is understood that the methods of this invention could be applied to provide insect protection, and more preferably coleopteran insect protection, to plant species from the genera Fabaceae, Medicago, Trifolium, Vigna, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Lycopersicon, Capsicum, Solanum, Nicotiana, Helianthus, Bromus, Asparagus, Panicum, Pennisetum, Cucumis, Lolium, Glycine, Triticum, Gossypium and Zea. In addition, forestry crop species from the genera Pinus, Populus, Eucalyptus, Acacia, Silex, and Larix are also prone to important coleopteran pest infestation which may be controlled by the methods and compositions described herein. Also, turf grass species such as St. Augustine (Stenotaphrum secundatum), Kentucky blue grass (Poa pratensis), and creeping bentgrass (Agrostis stolonifera) among others are susceptible to coleopteran pests such as white grub and the like which may also be controlled by the present invention. Insect pests which infest Roses (Rosa) and perennials such as Begonia, Pelagonium, Imaptiens, Tagetes, Viola, Petunia and Catharanthus and the like may also be subjects of the present invention.